In recent years, convenient examination reagents, diagnostic reagents and diagnostic kits have been developed for conducting various examinations for the presence of pathogenic infection by viruses, bacteria or the like, the presence or absence of pregnancy, the presence or absence of cancer markers, and the presence or absence of specific raw materials or hazardous substances such as residual pesticides in foods, in short periods of time. These utilize specific reactions between the different substances to be examined and substances that specifically react with the substances to be examined. In particular, a large number of assay methods have been developed as immunoassays using antigen-antibody reaction between antigens and antibodies, including immunochromatographic assays, turbidimetric immunoassays, enzyme immunoassays, chemiluminescent assays, radioimmunoassays, assay methods using surface plasmon resonance, and the like. These assay methods are also utilized for disease examination at hospitals, clinics and the like, and for food examination at food companies, for example. Among these, immunochromatographic assays do not require special equipment, devices or knowledge and their operation is convenient and economical, while they also allow rapid diagnosis, and therefore they are commonly implemented in a large range of examinations. Pregnancy examination agents, HIV examination agents and the like have come to be sold at ordinary pharmacies in recent years, allowing measurement by ordinary consumers, and it has also become possible to perform not only qualitative examinations for examining the presence or absence of substances to be examined, but also quantitative examinations for measurement of their amounts.
The principle of measurement in an immunochromatographic assay may be that of the “sandwich method” or of the “competitive method”. Moreover, the measurement system may be based on a flow-through method or a lateral flow method. It is possible to detect a variety of different substances as substances to be examined in a specimen, and a typical example is measurement by detection of antigen by the sandwich method, in which the following procedure is carried out in order.
(1) A antibody that specifically binds with an antigen as the substance to be examined is immobilized at a prescribed section of a chromatographic medium such as a nitrocellulose membrane, and a reaction site known as the “test line” (hereunder referred to as “TL”) is formed at a desired location in the chromatographic medium.
(2) A detection reagent is prepared by supporting an antibody that specifically binds with the substance to be examined, on a labeling substance such as an enzyme, chromogenic particles, fluorescent particles, magnetic particles or the like, and the detection reagent is coated and dried onto a conjugate pad or the like, a detection reagent-containing section is formed, and this is combined with the chromatographic medium to form an immunochromatographic diagnostic kit.
(3) The actual specimen containing the antigen, or a solution containing it diluted with a desired liquid, is dropped onto a prescribed location of the immunochromatographic diagnostic kit, such as onto the sample pad, and the antigen and the detection reagent are developed on the chromatographic medium.
Using this series of procedures, the labeling substance is captured via the antigen on the antibody immobilized on the chromatographic medium at the reaction site, and the labeling substance signal is detected for diagnosis by the immunochromatographic diagnostic kit. Ordinarily, diagnosis is qualitative diagnosis that detects only the presence or absence of the antigen, but in recent years the strength of the signal can be visually or mechanically detected for quantitative diagnosis.
A rapid diagnosis time is a requirement for an immunochromatographic assay. This is in order to shorten the waiting time for the examination. A common method for meeting this requirement is a method of adjusting the pore sizes of the chromatographic medium to increase the traveling speed of the specimen.
Also, PTL 1 reports that a more rapid diagnosis time is possible by using a specific cellulosic fiber nonwoven fabric as the sample pad, at the section where the specimen sample containing the substance to be examined is to be dropped. PTL 1 mentions the liquid absorption speed and developability, but contains no concrete data for the diagnosis time and nowhere mentions a combination with specific chromogenic particles, as according to the present invention. In addition, it does not mention modification to the placement of the sample pad and conjugate pad.
Furthermore, PTL 2 reports that it is possible to achieve a more rapid diagnosis time by modifying the structure of the diagnostic kit and the like, but the shortest assessment in a hCG assay, for example, is 2 minutes and 35 seconds.
Increased analysis sensitivity is another requirement for an immunochromatographic assay. This means the ability to accomplish detection with an even lower amount of substance to be examined. In PTL 3, the present inventors have reported that analysis sensitivity can be increased by using cellulose particles having dense color and large particle diameters as the chromogenic particles. However, while it is mentioned that rapid diagnosis is possible by using chromogenic particles having dense color and large particle diameters, there is no concrete data for the diagnosis time and nothing is mentioned regarding combination with a specific sample pad, as according to the present invention.
Thus, there is generally a trade-off between speed of diagnosis time and increased analysis sensitivity, and a very great need exists for simultaneously achieving both.
In addition, PTL 4 discloses a semiquantitative test method using immunochromatography, and states that the antigen concentration and examination result signal strength can be made proportional for antigen concentrations in certain ranges. However, for quantitation in immunochromatography it is ideal to not only have proportionality between antigen concentration and examination result signal strength at certain antigen concentrations, but also to have reproducibility of test results, i.e. to obtain a signal of the same intensity when measuring antigen of the same concentration, but it is the general consensus that such reproducibility is still inadequate.
Also, PTL 5 discloses an immunochromatographic diagnostic kit using fluorescent particles as chromogenic particles, mentioning cellulose particles as an example for the material for the chromogenic particles and a cellulose nonwoven fabric as an example of the material for the sample pad, but neither are described in specific detail. In addition, it does not mention modification to the placement of the sample pad and conjugate pad. Naturally, nothing is mentioned regarding an effect by combination of the sample pad and the chromogenic particles, as according to the present invention.